Embodiments of the present invention relate to flow cytometry, and in particular to systems and methods for assessing the quality of results obtained by boundary placement techniques.
Flow cytometry immunophenotyping of hematopoietic disorders is a complex and demanding task that requires a good understanding of cell lineages, developmental pathways, and physiological changes, as well as broad experience in hematopathology.
Flow cytometry allows simultaneous multiparametric analysis of thousands of particles per second by suspending cells in a stream of fluid and passing them by an electronic detection apparatus. The data generated can be plotted into histograms and divided into regions. Regions are shapes that are drawn or positioned around a population of interest on a one or two parameter histogram. Exemplary region shapes include two dimensional polygons, circles, ellipses, irregular shapes, or the like. Individual events exemplified in the data correspond to unique combinations of parameters, and are accumulated in cases where multiple instances of such combinations are present. When a region is used to limit or isolate cells or events that are drawn or positioned on a histogram, such that those isolated cells or events can be manifested in a subsequent histogram, this process is referred to as gating. The data accumulated into histograms can be separated or clustered based on fluorescence intensity, in a series of sequential steps known as “gating” involving one or more regions. In some cases, gates are combined with each other using Boolean logic (AND, OR, NOT). A common technique involves using gates sequentially. In some cases, gates are performed in parallel.
In the last decade, advances in instrumentation and reagent technologies have enabled simultaneous single-cell measurement of tens of surface and intracellular markers, as well as tens of signaling molecules, positioning flow cytometry to play an ever increasing role in medicine and systems biology.
However, the rapid expansion of flow cytometry applications has outpaced the functionality of traditional analysis tools used to interpret flow cytometry data such that scientists are faced with the daunting prospect of manually identifying interesting cell populations in 20 dimensional data from a collection of millions of cells.
The Beckman Coulter tetraCXP system software, stemCXP and CytoDiff CXP software are nonlimiting examples of automated flow cytometry solutions. They provide a gating algorithm to separate populations of interest in the multidimensional space and report percentages and other clinical parameters to the user. Automated identification of homogenous cell populations that share a particular function is referred to as automated gating. These solutions alleviate the need for high expertise and reduce the processing time required for manually gating flow cytometry data. Additional benefits include labor cost reductions, uniformity in the analysis, and reduction of user induced errors.
A 2005 study involving 15 institutions (H. T. Maecker, A. Rinfret, P. D'Souza, et al., “Standardization of cytokine flow cytometry assays,” BMC Immunology, vol. 6, article 13, 2005) showed that the mean interlaboratory coefficient of variation ranged from 17-44%, even though the same samples and reagents were used and the preparation of samples was standardized. Even though all analyses were conducted by individuals with expertise in flow cytometry, most of the variation was attributed to gating.
Hence, although flow cytometry assay technologies provide real benefits to the field of hematology, still further advances and improvements are desirable. Embodiments of the present invention provide solutions to at least some of these outstanding needs.